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Reproduction | Cooled Semen | Container Study

Container Study

Effect of ambient temperature and container on temperature of extended equine semen

Introduction

The first use of cooled transported equine semen was reported by Douglas-Hamilton et al in 1984. They developed and described a thermal insulated container that slowly cooled semen to ~ 50C and maintained the temperature for at least 24 hours. They reported that a cooling rate of -0.080C per minute was well tolerated by most stallions. Later others reported similar results. In the intervening period there has been a sustained interest in and increasing usage of cooled transported semen that has resulted in the development and marketing of a variety of disposable containers. The introduction of new disposable shipment containers has not always provided semen in a viable condition at delivery. Frustrated clients often blame poor motility on poor initial semen characteristics, semen extender interactions and poor on farm breeding techniques. Apparently little information is available as to the efficacy of these containers either by way of a comparison between containers or ability to handle different environmental conditions that may be encountered in transit.

For equine semen to maintain its viability over time it must be cooled slowly. Containers that cool semen by transferring heat (i.e. to a coolant can) are called passive cooling systems. They generally remove heat fast at the beginning and slowly when the target temperature is closer (Figure 1). Active cooling systems remove heat (usually with electricity) and are controlled (Figure 1). Recent experiments at Colorado State University have been useful in determining the optimum cooling rate that can be tolerated by most stallion spermatozoa (Graham, 1993). The results of these experiments using programmed cooling rates over defined temperatures have demonstrated that semen can be cooled quickly between 370C and 190C (0.70C/min) but that between 180C and 80C the extended semen needs to be cooled slowly (0.050C) to avoid cold shock.

The aim of our experiment was to compare the effect of volume and temperature on temperature of extended equine semen that was stored according to manufacturers recommendations in a variety of disposable containers designed for semen transport.

 

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Figure 1. Cooling rates for active (ideal) and passive cooling systems (Equitainer at room temperature).

 

Materials and methods.

Five containers specifically designed for slow cooling of equine semen were evaluated with semen prepared according to the manufactures recommendations. Semen was extended at 370C to a concentration 20 X 106/ml and a total volume of 100 ml, or 200 X 106/ml and a total volume of 10 ml and allowed to equilibrate slowly to room temperature (~ 220C) over ~ 15 minutes. After the addition of the extended semen each container was subjected to one of three environmental tempertures: a) Room temperature, b) Heated environment (placed inside a sealed car in the sun) or 3) Cool environment (placed inside a refrigerator). Temperature of the extended semen was recorded (thermocouple- accurate to ± 0.050C) without opening or moving the containers, hourly for the first 12 hours and then every three hours for the next 24 hours. Progressive motility was recorded at the beginning and end of replicate. Two replicates were performed for each volume (10 and 100 ml) for semen stored under the three environmental conditions. The experiment was performed in February in southern Australia.

 

Results

The environmental temperatures that the semen containers were subjected too can be visualized in Figure 2. Car temperatures exceeded 500C for hours during the hottest part of the day, however room temperature and the cool room were quite stable.

 

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Figure 2. Environmental temperatures recorded in the three different locations: a) Room temperature, b) Heated environment (placed inside a sealed car in the sun) or 3) Cool environment (placed inside a refrigerator).

 

The temperature responses of stored extended semen (100 ml) in each individual container subjected to the three environmental temperature are recorded in Figures 3-7. There were large variations of temperature of extended semen according to both container type and environmental conditions.

 

 

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Figure 3 Effect of environmental temperature on temperature of extended semen (100 ml) stored in an Equitainer.

 

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Figure 4 Effect of environmental temperature on temperature of extended semen (100 ml) stored in the Expecta Foal container.

 

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Figure 5 Effect of environmental temperature on temperature of extended semen (100 ml) stored in the Bio-Flite container.

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Figure 6 Effect of environmental temperature on temperature of extended semen (100 ml) stored in Lane STS container.

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Figure 7 Effect of environmental temperature on temperature of extended semen (100 ml) stored in the NZ Semen Shipper.

 

Individual responses of each container compared to the others can be visualized from Figures 8-10

 

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Figure 8 Effect of container on temperature of extended semen (100 ml) stored under different environmental conditions (Room temperature).

 

 

 

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Figure 9 Effect of container on temperature of extended semen (100 ml) stored under different environmental conditions (Car).

 

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Figure 10 Effect of container on temperature of extended semen (100 ml) stored under different environmental conditions (Refrigerator).

 

 

 

 

 

 

The effect of volume for each container was made by comparison of 10 ml versus 100 ml and results are graphed in Figures 11-15.

 

 

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Figure 11 Effect of extender volume on temperature for the Equitainer

 

 

 

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Figure 12 Effect of extender volume on temperature for the Expecta Foal

 

 

 

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Figure 13 Effect of extender volume on temperature for the Bio-Flite

 

 

 

 

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Figure 14 Effect of extender volume on temperature for the Lane STS

 

 

 

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Figure 15 Effect of extender volume on temperature for the NZ Semen Shipper

 

The effect of container, environmental temperature and volume of extended semen on progressive motility is presented in the Table below.

 

 

 

Car

Car

Room T

Room T

Cold

Cold

Motility

100 ml

10 ml

100 ml

10 ml

100 ml

10 ml

Equitainer

57.5

50

52.5

60

55

55

Bio-Flite

45

5

50

50

55

45

Lane STS

37.5

42.5

42.5

40

30

35

Expecta Foal

25

0

42.5

20

30

35

NZ Semen Shipper

12.5

0

52.5

35

55

40

Table 1. Effect of storage conditions and containers on progressive motility of equine semen.

 

Discussion

The results of this experiment suggest that some containers have more uniform cooling rates and stability than others and that environmental conditions have an important role in the ability of the containers to accurately cool extended semen. If containers are placed in areas such as cargo holds in buses they may become warm and conversely they may become cold in cargo holds of planes (McKinnon, 1996). The interplay of outside temperature and cooling container has enormous impact on the semen cooling rates and thus motility.

None of the containers performed well in the hot conditions. There was a wide variation from the ideal cooling rate with most containers, however with the exception of the Expecta Foal most performed well at room temperature for 20-24 hours.

This experiment suggests that larger volumes appear to be less susceptible to variations in outside temperature and in higher temperatures large volumes tended to heat slower, reach lower maximum temperatures, cool quicker and maintain better progressive motility than small volumes.

 

References.

 

Douglas-Hamilton, D.H., Osol, R., Osol, G., Driscoll, D., and Noble, H. A field study of the fertility of transported equine semen. Theriogenology 22:291-304, 1984.

 

Graham, J.K. Biology and structure or spermatozoa, and their response to cooling. Proceedings: Techniques for handling and utilization of transported cooled and frozen equine spermatozoa. Equine Sciences Program, Colorado State University, 8-21, 1993.

 

McKinnon, A.O. Artificial insemination of cooled, transported and frozen semen. In: Equine Stud Medicine, Sydney: Post Graduate Foundation in Veterinary Science,. 319-337, 1996

 

Acknowledgments:- We would like to thank Meagan Strickland-Wood and Fiona Napier for their assistance in data recording.